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Journal of Cereal Science 26 (1997) 37–46

Determination of the Pentosan Content of Wheat


Products by Hydrolysis, Glucose Oxidase
Treatment and Analysis by HPAEC/PAD
R. Houben, C. F. de Ruijter and K. Brunt

Netherlands Institute for Carbohydrate Research TNO, Rouaanstraat 27,


9723 CC Groningen, The Netherlands

Received 28 May 1996

ABSTRACT
A method was developed for determining routinely the total pentosan content of wheat flour,
commercial B-starch and gluten. In this method, the polysaccharides in the sample were hydrolysed
by treatment with 2 HCl at 100°C. The resulting pentosan monosaccharides were characterised
by anion-exchange chromatography (HPAEC) combined with pulsed amperometric detection (PAD).
Using this method, water-extractable pentosans can be determined with only minor adjustments.
Interference by the relatively high concentration of starch-derived glucose in the sample, in the
HPAEC separation of the pentosan monosaccharides, was eliminated by enzymic conversion of
glucose into gluconic acid using glucose oxidase (GOD). Efficient removal of glucose (1 h incubation
time) required a relatively high amount of GOD and a continuous supply of oxygen into the solution.
The activity of GOD against galactose and xylose was subsequently blocked by lowering the pH of
the sample to about 1. The method described has good repeatability. The standard deviation in the
pentosan content (sum of the pentose sugars arabinose and xylose) was less than 0·1% for flour, B-
starch (pentosan content about 2%) and gluten (pentosan content of about 1%).
 1997 Academic Press Limited

Keywords: pentosan, monosaccharides, content, wheat, HPAEC/PAD, GOD.

INTRODUCTION binoxylan, with a linear xylan backbone and a


high degree of branching with single arabinose
Pentosan constitutes a major part of the non- side residues and arabinogalactan, with a galactan
starch polysaccharides in wheat flour, with a total backbone and a high degree of branching with
pentosan content of about 2%1–3. The water-ex- arabinose side residues6.
tractable fraction of pentosan makes up between The pentosan content in wheat flour can be
0·5 and 0·8%1,2. Water-extractable pentosan may
determined by colorimetric methods, such as the
absorb up to nine times its own weight of water
orcinol-HCl2,7 and the phloroglucinol8–10 methods.
and water-unextractable pentosan up to five times
However, these methods are interfered with
its own weight4. The high water-binding capacity
by starch-derived glucose. Furthermore, no in-
of pentosan reduces the availability of water for
formation is obtained about the sugar composition
starch gelatinisation5. Pentosans consist of ara-
of the pentosan. The pentosan content can also
be determined by gas chromatography, following
hydrolysis and dehydration of pentosan sugars to
 : GOD = glucose oxidase; furfural and extraction with dibutylether11.
HPAEC = high performance anion-exchange However, hexose sugars, which form 5-hydroxy-
chromatography; PAD = pulsed amperometric de- methylfurfural, interfere with the selective ex-
tection. traction of furfural. Non-starch polysaccharides

0733–5210/97/040037+10 $25.00/0/jc960110  1997 Academic Press Limited


38 R. Houben et al.

can also be determined by liquid chromatography in the tube was not removed. Following hydrolysis,
following hydrolysis12 or by gas chromatography, the sample was cooled and subsequently neu-
following hydrolysis and derivatisation to alditol tralised by addition of 4·0  NaOH (2·0 mL). Excess
acetates13 or to trimethylsilyl esters14. To prevent glucose was removed by addition of glucose ox-
interference of glucose, starch can be enzymically idase/catalase solution (2·0 mL) in 2·0  acetate
removed prior to hydrolysis12,13. buffer and 60 min incubation at room temperature.
In our study, a method was developed for de- During incubation the sample was mixed con-
termining routinely the pentosan content in wheat tinuously with the air present in the tube to en-
flour, commercial B-starch and gluten by anion- hance dissolution of oxygen. Finally the pH of the
exchange chromatography (HPAEC) with pulsed solution was lowered by addition of 4·0  HCl
amperometric detection (PAD). In this method, (1·0 mL) and an aliquot (0·5 mL) of internal stand-
the interference of glucose was eliminated by con- ard was added (1 mg/mL of 2-deoxy-D-galactose
version of glucose to gluconic acid with the enzyme in 0·04  HCl).
glucose oxidase (GOD). The method of analysis The amount of water-extractable pentosan can
is advantageous, because sample pretreatment is also be determined; this requires only minor ad-
simple and no derivatisation is required while the justments. The sample (ca. 0·4 g) was extracted
method gives both quantitative and qualitative with water (8·0 mL) for 2 h at 30°C2. After cen-
information about pentosan. trifugation, an aliquot (2·0 mL) of the supernatant
was removed, 4·0  HCl (2·0 mL) was added and
MATERIALS AND METHODS the sample was hydrolysed as described before.

Chemicals
Sample pretreatment was performed using hy- High performance anion-exchange
drochloric acid (Merck, Germany) and 50% (w/ chromatography
w) sodium hydroxide (Baker, Netherlands). For
the removal of glucose, the enzymes glucose ox- Samples were injected using a Marathon auto-
idase (EC 1.1.3.4, grade II, Boehringer, Germany) sampler (Spark, Holland) with a Tefzel rotor and
and catalase (EC 1.11.1.6, Sigma, U.S.A.) were a 20 lL loop. The pumps were obtained from
used, dissolved in buffer at 1000 U/mL and 6500 Gilson (France). The monosaccharides were sep-
U/mL, respectively. The buffer solutions were arated using a CarboPac PA1 precolumn and
prepared from sodium acetate (Merck) and acetic a CarboPac PA1 analytical column (Dionex,
acid (Merck) or tris(hydroxymethyl)-amino- U.S.A.). The monosaccharides were separated iso-
methane (Merck) and hydrochloric acid (Merck), cratically with 0·001  NaOH (45 min, flowrate
respectively. The following monosaccharides were 1 mL/min). Afterwards, strongly retained anions
used: galactose (Aldrich, Germany), glucose and were removed from the column by increasing the
1,5-gluconolactone (BDH, U.K.), xylose (Merck) NaOH concentration to 0·5  over a period of
and arabinose and 2-deoxy-D-galactose (Sigma). 6 min and continuing the elution with 0·5  NaOH
Wheat flour (Friso) was obtained from a local for a further 6 min. Finally, the NaOH con-
supermarket, while wheat B-starch (Puramyl VEM centration was reduced to 0·001  NaOH over a
II) was obtained from Latenstein (Netherlands). period of 1 min and the column was re-equilibrated
Wheat gluten was fractionated in our institute on with 0·001  NaOH for 7 min (flour and B-starch)
a laboratory scale. or for 4 min (gluten) before injecting another
sample. To maintain a high detector sensitivity,
0·25  NaOH was added to the effluent post-
Sample preparation prior to
column at a flow rate of 0·4 mL/min.
HPAEC/PAD analysis Monosaccharides were detected using a Dionex
For determination of the total pentosan content, PED I detector with a 3 mm gold working elec-
the sample (ca. 0·05 g) was dispersed in water trode. The potential of the electrode was pro-
(2·0 mL) and 4·0  HCl (2·0 mL) and then hy- grammed from +0·15 V (t = 0–0·72 s) to +0·7
drolysed for 90 min at 100°C in a 50 mL screw- V (t = 0·73–0·84 s) and finally −0·3 V (t =
cap tube. During hydrolysis, the samples were 0·85–1·2 s), while integrating the resulting signal
mixed frequently to aid dispersion. The air present from t = 0·52–0·72 s.
Determination of pentosan by HPAEC/PAD 39

2.50

2.00
Sugar content (% w/w)

1.50

1.00

0.50

0 30 60 90 120 150 180


Time of hydrolysis (min)

Figure 1 Content (weight % of the sample as is) of arabinose (+), galactose (Α) and xylose (Β) and sum of the pentose
sugars arabinose and xylose (Φ) – as a function of the time of hydrolysis. The wheat flour sample was hydrolysed in 2  HCl,
treated with GOD and analysed by HPAEC/PAD.

RESULTS AND DISCUSSION sugar content determined in the wheat flour


sample. The measured levels of pentosan mono-
Optimisation of the conditions for hydrolysis saccharides depend upon two simultaneous pro-
The highest yield of non-starch polysaccharides is cesses: hydrolysis of pentosan polysaccharides and
commonly obtained by hydrolysis with sulphuric relatively slow acid decomposition of the pentosan
acid15. A major disadvantage of sulphuric acid is monosaccharides formed16,17. Figure 1 shows that
the difficulty of removing the acid after sample 90 min of hydrolysis yielded good results; the
hydrolysis. Comparable yields can be obtained by polysaccharides were hydrolysed completely, while
using trifluoroacetic acid, which can be removed the decomposition of monosaccharides was still
by evaporation15,16. However, hydrolysis with HCl relatively low. Figure 2 shows the acid de-
was preferred because the evaporation step is time- composition of a solution of monosaccharides as
consuming. We found that the effect of Cl− ions on a function of the time of hydrolysis. From this it
the retention of monosaccharides on the CarboPac was deduced that the recovery of monosaccharides
PA1 column was negligible after neutralisation after 90 min of hydrolysis with 2  HCl is about
and dilution of the sample. 85% for arabinose and galactose and about 75%
The conditions for hydrolysis of pentosan were for xylose. The loss of monosaccharides in the
derived from literature2. To determine the opti- determination of the pentosan content is probably
mum time of hydrolysis, a wheat flour sample less because the monosaccharides have to be lib-
was hydrolysed with 2  HCl at 100°C for times erated from the pentosans before being degraded.
ranging from 0 to 180 min. Figure 1 shows the The recovery of monosaccharides after addition
40 R. Houben et al.

100

80

60
Recovery (%)

40

20

0 30 60 90 120 150 180 210


Time of hydrolysis (min)

Figure 2 Recovery (%) of arabinose (+), galactose (Α), xylose (Β) and 2-deoxy-D-galactose (Φ) after hydrolysis and analysis
by HPAEC/PAD. The recovery is defined as the concentration of the monosaccharides determined after acid treatment
divided by the concentration of the mixture without acid treatment.

of a monosaccharide standard to wheat flour was Elimination of glucose interference by treatment


comparable to or even slightly higher than deduced with glucose oxidase
from Figure 2 (respectively, 90% for arabinose,
85% for galactose and 85% for xylose). Therefore, The bulk of wheat flour consists of starch (about
it can be concluded that no extra loss of mono- 80%), with pentosan as a minor component (about
saccharides occurred because of reaction with the 2%). In the hydrolysed sample, the concentration
sample matrix. of glucose greatly exceeds the concentration of
From Figure 2 it can be concluded that 2-deoxy- pentosan sugars, and this interferes with the
D-galactose cannot be used as an internal standard HPAEC/PAD analysis. Therefore, we used the
for the entire analytical procedure because of the enzyme glucose oxidase (GOD) to convert glucose
rapid acid decomposition of this sugar (complete into gluconic acid, which is retained much more
decomposition in 30 min of hydrolysis). Therefore, strongly by the HPAEC column. The GOD treat-
in our method the internal standard was added ment resulted in a greatly diminished glucose peak,
to the sample following hydrolysis. The internal which no longer interfered with the chro-
standard was used only to compensate for fluc- matographic separation of the pentosan mono-
tuations in PAD sensitivity. saccharides (Fig. 3). Compared with methods such
Determination of pentosan by HPAEC/PAD 41

52.84
800.00

600.00
Integrator signal (mV)

400.00

4
23.62

200.00

1
2
5
9.61

14.92

3
28.48
20.17

0.00

0 10 20 30 40 50 60
Time (min)

Figure 3 HPAEC/PAD chromatogram of a wheat flour sample after hydrolysis and GOD-treatment. The chromatographic
peaks can be assigned to (1) 2-deoxy-D-galactose (internal standard), (2) arabinose, (3) galactose, (4) glucose, (5) xylose and (6)
gluconic acid.

as those described in the literature by Englyst et consumed18. At a high glucose concentration, the
al.12,13, no time-consuming sample pre-treatment is level of available oxygen may be limiting. This
required consisting of enzymic starch degradation can, in part, be overcome by adding catalase which
followed by precipitation of pentosans with al- converts the hydrogen peroxide formed during the
cohol. GOD reaction into water and oxygen. Fur-
In the enzymic conversion of b-D-glucose, D- thermore, as a result of adding catalase, inhibition
gluconic acid is formed and molecular oxygen is of GOD by hydrogen peroxide is prevented. How-
42 R. Houben et al.

100

90

80
Degree of conversion (%)

70

60

50

40
15 30 45 60
Incubation time (min)

Figure 4 Degree of conversion of glucose (100 mg) when incubated with, respectively, 8000 U (+), 6000 U (Α), 4000 U
(Β) and 2000 U (Φ) of GOD in a large volume tube with a constant supply of air. When the sample was incubated in a
small volume tube (with 8000 U GOD), oxygen deficiency occurred (Η).

ever, only half the amount of oxygen consumed approximately 540 mg of glucose/min at pH 8·0
in the GOD conversion of glucose can be regained. (according to specifications).
We concluded that oxygen may become limiting
if incubation is carried out in closed tubes (Fig.
Quantification of the pentosan content
4). The oxygen consumed in the conversion of
glucose can be replenished readily, however, by A well-known problem in electrochemical de-
incubating in screw cap tubes with a relatively tection is the relative instability of the detector
large volume (4 mL of sample in a 50 mL tube), due to electrode fouling. This is partly overcome
continuously mixing the solution with air present in pulsed amperometric detection by programming
in the tube and frequently opening the tube to the potential of the electrode in a continuous way19.
refresh the air present. Nevertheless, detector sensitivity may change a
The rate of glucose conversion with GOD was little in the course of time, and this necessitates
much lower than expected. In a control ex- the use of an internal standard. In our study, we
periment, glucose (0·1 g) in Tris-HCl buffer, pH tested the sugars 2-deoxy-D-glucose, fucose and 2-
8·0 was treated with different amounts of GOD deoxy-D-galactose as internal standard. We found
and for different incubation times. As can be seen that 2-deoxy-D-glucose was not suitable because
in Figure 4, complete conversion of glucose within it was not well resolved from rhamnose, which
60 min required more than 4000 U of GOD. may be present in the sample in a low con-
This amount of enzyme, however, should convert centration. Fucose was not suitable because its
Determination of pentosan by HPAEC/PAD 43

Table I Recovery of the monosaccharides 2-deoxy-D-galactose (deoxygal), arabinose (ara),


galactose (gal), glucose (glc) and xylose (xyl) after treatment with GOD in acetate buffer
(pH=5)

Incubation deoxygal (%) ara (%) gal (%) glc (%) xyl (%)
time (h)

1 101 101 98 5 97
2 98 100 93 2 92
3 97 99 90 0 90
4 96 98 84 0 87
24 98 101 32 0 44

The concentration of the monosaccharides in the test mixture (about 50 mg/L) and the
amount of GOD added (2000 U) were comparable to the conditions described in the Materials
and Methods section.

chromatographic peak coincides partly with PAD- preserve the sample for a relatively long period of
sensitive compounds in the sample solution time. In this way, no (enzymic or acid) degradation
(e.g. Tris-buffer). These compounds temporarily of pentosan monosaccharides was observed over
lowered the detector sensitivity and thus interfered a time period of 7 days.
with the quantification of the internal standard.
2-Deoxy-D-galactose was finally chosen as the
internal standard because this sugar does not occur
naturally in wheat and it was well resolved from Determination of the pentosan content
pentosan monosaccharides (Fig. 3). It was added of wheat gluten
after hydrolysis because of the rapid degradation For the determination of the pentosan content of
of this sugar under acidic conditions (Fig. 2). wheat gluten, a small adjustment of the method
was necessary. As can be seen in Figure 5(a),
the detector signal was temporarily diminished
Substrate specificity of GOD
between retention times of about 6 and 9 min,
GOD has high specificity for b-D-glucose, but also probably due to a component resulting from pro-
catalyses the oxidation of galactose and xylose tein hydrolysis. This problem was negligible for
(relative rate about 100 times lower)18. The GOD wheat flour and wheat B-starch because of a much
incubation of the hydrolysed wheat sample should lower protein content. The disturbance in the
therefore be as short as possible, while nevertheless chromatographic signal partly coincided with the
ensuring almost complete conversion of glucose. chromatographic peak of 2-deoxy-D-galactose and
The effect of GOD on the pentosan sugars was thus hampered accurate quantification of the in-
determined by treating a mixture of mono- ternal standard. This interference led to a sig-
saccharides with GOD under the same conditions nificant over-estimation of the pentosan content.
as wheat samples (acetate buffer, pH=5) for time The problem was overcome by a small change
periods ranging from 1 to 24 h. The recoveries of in the chromatographic conditions, namely by
monosaccharides after GOD treatment are pre- lowering the interval between two injections from
sented in Table I. 7 to 4 min in combination with 1:1 dilution of the
From Table I it can be concluded that GOD sample with water prior to HPAEC/PAD analysis
catalyses the oxidation not only of glucose but also [Fig. 5(b)].
of galactose and xylose, whilst arabinose and 2- The recovery of arabinose and galactose, added
deoxy-D-galactose (internal standard) are hardly to the gluten matrix, and subsequently hydrolysed
affected. Treatment with GOD for 1 h resulted in with 2  HCl and analysed by HPAEC/PAD was
almost complete conversion of glucose to gluconic comparable to the recovery for a standard of
acid with negligible oxidation of pentosan mono- monosaccharides without sample. The recovery
saccharides. Subsequently, GOD activity needs to for xylose added to gluten, however, was about
be blocked by adding a small amount of HCl and 10% lower than when adding xylose either to
thus lowering the pH of the sample to about 1, to wheat flour or to wheat B-starch, implying that
44 R. Houben et al.

500.00

400.00
Integrator signal (mV)

3
300.00 1 2 18.97 4
9.08

14.10

22.17
5

26.44
200.00

100.00

(a)

0 5 10 15 20 25 30 35 40 45 50
Time (min)

500.00

400.00
Integrator signal (mV)

300.00 1
8.89

2 3
13.51

4
18.45

5
21.44

200.00
25.60

100.00

(b)

0 5 10 15 20 25 30 35 40 45 50
Time (min)
Determination of pentosan by HPAEC/PAD 45

Table II Standard deviation for the repeatability (sr) calculated from one-way analysis
of variance (ANOVA) from duplicate measurements on eight different days

Sample ara (%) gal (%) xyl (%) pentosan (%)

Flour content 0·91 0·38 1·18 1·84


standard deviation of 0·03 0·01 0·05 0·07
repeatability sr
B-starch content 0·80 0·13 1·21 1·77
standard deviation of 0·02 0·01 0·04 0·04
repeatability sr
Gluten content 0·55 0·77 0·53 0·95
standard deviation of 0·03 0·03 0·05 0·05
repeatability sr

The average contents of arabinose (ara), galactose (gal), xylose (xyl) and pentosan
(sum of pentose sugars arabinose and xylose multiplied by 0·88 to correct for anhydro
monosaccharides) are presented as weight % of the sample (as is).

xylose may be partly protected by the flour or B- resulting pentosan monosaccharides by anion-ex-
starch matrix. change chromatography (HPAEC) combined with
pulsed amperometric detection (PAD). The in-
terference of starch-derived glucose in the
Repeatability of the presented method HPAEC/PAD analysis can be eliminated simply
of analysis by enzymic conversion of glucose to gluconic acid
The standard deviation for the repeatability was with glucose oxidase. Following conversion of gluc-
determined by analysing the three sample matrices ose, the enzyme activity is blocked by lowering
in duplicate on eight different days. Table II the pH of the sample to about 1. The proposed
presents the standard deviation for the re- method for routine determination of the pentosan
peatability, as calculated by one-way analysis of content has good repeatability.
variance (ANOVA), and the calculated average
content. The standard deviation for the individual
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(b) with 4 min interval and 1:1 dilution. The chromatographic peaks are assigned to (1) 2-deoxy-D-galactose (internal standard),
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